Protozoan rhomboid proteins

ABSTRACT

Protozoan Rhomboid Proteins This invention relates to the identification of protozoan Rhomboid proteins that are involved in the invasive processes of protozoan pathogens, such as  P. falciparum.  Modulation of these Rhomboid proteins may thus be useful in treating protozoan pathogen infection. Methods and means relating to the modulation of protozoan Rhomboid proteins is provided herein.

The present invention relates to the invasive processes of protozoanpathogens and in particular to the provision of therapeutic compoundswhich block these processes and may thus be useful in treating protozoanpathogen infection.

Protozoan pathogens of the Apicomplexa family, which include the malariaparasite P. falciparum, express a number of membrane-tethered adhesionproteins that are essential for the recognition and binding of host(e.g. mammalian) cells. An essential step in the invasive processes bywhich these pathogens enter host cells is the release of these adhesionproteins from the surface of the pathogen by proteolytic cleavage. Someof these adhesion proteins are cleaved within the transmembrane domain(TMD) (Opitz et al (2002) EMBO J. 21 7 1577-1585).

The present inventors have discovered that Rhomboid polypeptides areinvolved in the cleavage of protozoan adhesion molecules. Rhomboidpolypeptides are intra-membrane serine proteases, which are widelyconserved throughout evolution and act on a range of physiologicalsubstrates. Inhibition of the protozoan Rhomboid activity reducesadhesion polypeptide cleavage and may thus reduce the invasiveness ofthe protozoan pathogen.

A first aspect of the invention provides a method for identifying and/orobtaining a compound that inhibits invasiveness of a protozoan pathogen,for example by inhibiting the activity of a protozoan Rhomboidpolypeptide, which method comprises:

(a) bringing into contact an isolated Rhomboid polypeptide and anisolated substrate polypeptide in the presence of a test compound; and

(b) determining proteolytic cleavage of the substrate polypeptide.

The Rhomboid and substrate polypeptides may be contacted out underconditions in which the Rhomboid polypeptide normally catalysesproteolytic cleavage of the substrate polypeptide.

The polypeptides may be contacted in a reaction medium in an isolatedform or may be comprised in a liposome or host cell, preferably, a hostcell in which the Rhomboid polypeptide and substrate are not naturallyexpressed. The Rhomboid polypeptide may, for example, act on amembrane-bound substrate polypeptide to generate a soluble product,which is detected.

Cleavage of the substrate polypeptide may be determined in the presenceand absence of test compound. A reduction or decrease in cleavage in thepresence of the test compound relative to the absence of test compoundmay be indicative of the test compound being an inhibitor of protozoanRhomboid protease activity. Such a compound may inhibit adhesivemicronemal polypeptide cleavage and thus protozoan pathogen infectivity.

A Rhomboid polypeptide may be a protozoan Rhomboid protein, for examplea Rhomboid polypeptide from an apicomplexan pathogen. Suitable protozoanRhomboid polypeptides include any one of Rhomboids 1-7 of P. falciparumas shown in Table 1a.

In some preferred embodiments, the Rhomboid polypeptide may be anon-mitochondrial Rhomboid, such as Rho1, Rho3, Rho4, Rho6 or Rho7 of P.falciparum, in particular Rho1, Rho3, Rho4 or Rho7.

Amino acid residues of Rhomboid and substrate polypeptides are describedin the present application with reference to their position in theDrosophila Rhomboid-1 and Spitz sequences, respectively. It will beappreciated that the equivalent residues in other Rhomboid and substratepolypeptides may have a different position and number, because ofdifferences in the amino acid sequence of each polypeptide. Thesedifferences may occur, for example, through variations in the length ofthe N terminal domain. Equivalent residues in other Rhomboid andsubstrate polypeptides are easily recognisable by their overall sequencecontext and by their positions with respect to the TMDs.

A polypeptide which is a member of the Rhomboid family preferablycomprises residues R152, G215, S217 and H281, more preferably residuesW151, R152, N169, G215, S217 and H281. The presence of these conservedresidues may be used to identify Rhomboid polypeptides in otherprotozoan pathogens.

Preferably, a Rhomboid polypeptide comprises at least 4 TMDs, morepreferably at least 5 TMDs, with residues N169, S217 and H281 eachoccurring in different TMD at about the same level in the lipid membranebilayer.

Preferred Rhomboid polypeptides comprise a GxSx motif, for example GxSG.

A Rhomboid polypeptide may also comprise additional amino acid residueswhich are heterologous to the Rhomboid sequence. For example, a Rhomboidpolypeptide or fragment thereof may be included as part of a fusionprotein, e.g. including a binding portion for a different ligand.

Whilst Rhomboid polypeptides may share relatively low overall sequenceidentity, they are reliably identified by bioinformatics techniques andmanual inspection of key residues, as described herein.

A polypeptide which is a member of the Rhomboid family (i.e. a Rhomboidpolypeptide) may be identified by the presence of a Rhomboid homologydomain, as defined by the PFAM protein structure annotation project(Bateman A. et al (2000) The Pfam Protein Families Database Nucl. Acid.Res. 28 263-266). The Pfam rhomboid homology domain is built from aHidden Markov Model (HMM) using 83 rhomboid sequences as a seed. ThePfam ‘rhomboid’ domain has the pfam specific accession number PF01694.

Various other methods suitable for use in identifying Rhomboidpolypeptides are known in the art.

Particularly valuable methods include the use of Hidden Markov Modelsbuilt from groups of previously identified Rhomboid proteins, including,but not limited to Drosophila Rhomboids 1-4. Such bio-informaticstechniques are well known to those skilled in the art (Eddy S. R. Curr.Opin. Struct. Biol. 1996 6(3) 361-365).

Rhomboid polypeptides are preferably able to proteolytically cleave oneor more of Drosophila proteins Spitz, Keren and Gurken or protozoanproteins such as Amal and CTRP, but not similar type I transmembraneproteins like TGFA, Delta, EGFR, and TGN38.

Suitable substrate polypeptides are Type 1 membrane proteins with asingle TMD orientated with an N terminal extracellular/lumenal domain. Asubstrate polypeptide may comprise a transmembrane domain which has alumen proximal region having one or more of the residues of residues138-144 of the Drosophila Spitz sequence (ASIASGA), or a lumenal regionhaving an equivalent conformation, structure or three dimensionalarrangement to that of residues 138-144 of the Drosophila Spitz sequence(ASIASGA).

In preferred embodiments, residues 1 to 7 of the TMD of a substratepolypeptide are not hydrophobic.

A substrate polypeptide may comprise a G residue within the portion ofthe TMD proximal to the lumenal or extracellular domain of thepolypeptide (i.e. between residues 1 and 8 of the TMD), preferably, theG residue being at a position equivalent to Spitz G143 (i.e. the 6^(th)residue of the TMD). Preferably, a substrate polypeptide comprises a GAor GG motif within the portion of the TMD proximal to the lumenal orextracellular domain of the polypeptide (i.e. between residues 1 and 8of the TMD), preferably at positions equivalent to G143 and A144 of theSpitz sequence (i.e the 6^(th) and 7^(th) residues of the TMD).

A substrate polypeptide may further comprise a triplet motif of smallamino acid residues at positions equivalent to A138, S139 and I140 ofSpitz (i.e. the 1^(st), 2^(nd) and 3^(rd) residues of the TMD asnumbered from the N terminal (extracellular) boundary of the TMD). Smallresidues include Gly, Ala, Ser, Ile, Leu, Val and Thr. Suitable motifsat this position include motifs which have Ala at a position equivalentto Spitz A138, such as AGG and ASI. The presence of Phe at positions 1,2 or 3 of the TMD of a polypeptide is a counter-indication for thepolypeptide being a Rhomboid substrate (i.e. such a polypeptide isunlikely to be efficiently cleaved by a Rhomboid polypeptide).

Suitable substrate polypeptides may include protozoan adhesivemicronemal polypeptides. An adhesive micronemal polypeptide may be froma protozoan pathogen, for example an Apicomplexan pathogen. Suitableadhesive micronemal polypeptides include transmembrane proteins thatbelong to the thrombospondin-related-anonymous proteins (TRAPs) family.

A protozoan adhesive micronemal polypeptide which is a suitablesubstrate may include an AGG or AGL motif at residues 1, 2 and 3 of theTMD as described above and a GG motif at positions 6 and 7 of the TMD.The protozoan pathogen may a member of the Apicomplexa family, forexample an apicomplexan pathogen selected from the group consisting ofPlasmodium, Toxoplasma, Eimeria, Sarcocystis, Babesia, Isospora,Cyclospora and Cryptosporidium, for example P. falciparum, P. bergei, T.gondii, C. parvum, E. tenella, S. muris, Babesia bovis, Cyclosporabelli, Theileria annulata or Theileria parva.

Examples of suitable polypeptides include AMA1, MIC2, MIC6, MIC8, MIC12,(from T. gondii), AMA1, TRAP, CTRP (from P. falciparum and P. berghei),MIC1 and MIC4 (from E. tenella) or other polypeptide as shown in Table2.

In some preferred embodiments, the substrate polypeptide may be CTRP orAMA1, for example P. falciparum CTRP or AMA1

The substrate polypeptide and the Rhomboid polypeptide may each comprisean ER (endoplasmic reticulum) retention signal. For example, a rhomboidpolypeptide may comprise a C terminal (lumenal) KDEL motif and asubstrate may comprise a C terminal (cytoplasmic) KKXX motif (Jackson etal (1993) J. Cell Biol. 121(2) 317-333)

The substrate polypeptide may comprise a detectable label, 20 such asgreen fluorescent protein (GFP), luciferase or alkaline phosphatase.This is preferably located in the soluble extracellular domain, allowingconvenient detection of the soluble cleaved product and is particularlyuseful in automated assays.

Methods for obtaining or identifying modulators, in particularinhibitors, of protozoan pathogen infectivity may be cell-based ornon-cell-based.

In non-cell based methods, the rhomboid polypeptide and the substratepolypeptide may be isolated or contained in a liposome. Liposome basedassays may be carried out using methods well-known in the art (BrennerC. et al (2000) Meths in Enzymol. 322 243-252, Peters et al (2000)Biotechniques 28 1214-1219, Puglielli, H. and Hirschberg C. (1999) J.Biol. Chem. 274 35596-35600, Ramjeesingh, M. (1999) Meths in Enzymol.294 227-246).

Preferably, methods according to the present invention take the form ofcell based methods. A cell based method may be performed in a cell suchas a protozoan cell (e.g. a Toxoplasma cell), a yeast strain, insect ormammalian cell line such as CHO, HeLa and COS cells, in which therelevant polypeptides or peptides are expressed from one or more vectorsintroduced into the cell.

In a preferred embodiment, the Rhomboid polypeptide and the substratepolypeptide may be expressed in a host cell from heterogeneous encodingnucleic acid.

Nucleic acid encoding the Rhomboid polypeptide and the substratepolypeptide may be contained on a single expression vector or onseparate expression vectors.

Persons skilled in the art may vary the precise format of methods of theinvention using routine skill and knowledge. For example, in someembodiments, the cleaved GFP moiety of a GFP-substrate fusion may becaptured with an anti-GFT antibody (Santa Cruz Biologicals), washed andthen the captured GFP detected with a polyclonal or monoclonal antibodyconjugated to an enzyme (capture ELISA) or a fluorescent label.

Some embodiments may employ an ELISA format in which, for example, asuitable polyclonal anti-GFP conjugated to horse-radish peroxidase or toalkaline phosphatase may be used. Such a conjugate is preferred sincethe number of incubations required is reduced. Alternatively, abiotinylated anti-GFP antibody in combination with an avidin orstreptavidin enzyme conjugate may be used.

In other embodiments, fluorescence detection may be used, for exampleusing a Europium- or Terbium-labelled antibody or streptavidin e.g.Delphia or Lance reagents, Perkin Elmer). These labels show a longfluorescence lifetime and have improved signal:noise ratiocharacteristics.

In other embodiments, GFP may be replaced in the GFP-substrate constructwith a different enzyme label at the N-terminus to give a direct assayfor the cleaved substrate in the medium (or the label may be added tosuch a construct). Suitable enzymes include Renilla luciferase (Lui, J.,and Escher, A. (1999) Gene 237, 153-159) and secretable alkalinephosphatase sequence (SEAP) (Clontech).

It is not necessary to use the entire full-length proteins for in vitroor in vivo assays of the invention. Polypeptide fragments as describedherein which retain the activity of the full length protein may begenerated and used in any suitable way known to those of skill in theart. Suitable ways of generating fragments include, but are not limitedto, recombinant expression of a fragment from encoding DNA. Suchfragments may be generated by taking encoding DNA, identifying suitablerestriction enzyme recognition sites either side of the portion to beexpressed, and cutting out said portion from the DNA. The portion maythen be operably linked to a suitable promoter in a standardcommercially available expression system. Another recombinant approachis to amplify the relevant portion of the DNA with suitable PCR primers.Small fragments (e.g. up to about 20 or 30 amino acids) may also begenerated using peptide synthesis methods, which are well known in theart. Another approach is to synthesise a nucleic acid comprising all orpart of the coding sequence from a series of synthetic oligonucleotides.The coding sequence may be synthesised using a more optimal genetic codefor the host cells (Kocken C H et al Infect Immun. (2002) 70(8):4471-6), for example reflecting mammalian codon usage for expression inmammalian cells.

A Rhomboid polypeptide fragment may consist of fewer amino acid residuesthan said full-length polypeptide. Such a fragment may consist of 325amino acids or less, 300 amino acids or less, or 275 amino acids or lessand/or may consist of at least 100 amino acids, more preferably at least150, 200, 250 or 300 amino acids. A suitable fragment may comprise fiveTMDs.

Such a fragment preferably comprises residues equivalent to R152, G215,S217 and H281, more preferably residues W151, R152, N169, G215, S217 andH281 of the Drosophila Rhomboid-1 sequence (Acc No: P20350), which areimportant for the catalytic activity of the protein and are highlyconserved in the Rhomboid family.

A substrate polypeptide fragment comprises fewer residues than thefull-length polypeptide and preferably comprises the transmembranedomain of the polypeptide. The TMD may be conveniently identified usingcommercially available software such as TMHMM (Krogh A. et al (2001) J.Mol. Biol. 305 567-580) and TmPred (Hofmann K & Stoffel W (1993) BiolChem. Hoppe Seyler 374 166).

In some embodiments, a chimeric substrate polypeptide may be used whichcomprises a substrate polypeptide TMD which is cleavable by a Rhomboidpolypeptide and heterogeneous intra- and extra-cellular domains.

Combinatorial library technology (Schultz, J S (1996) Biotechnol. Prog.12: 729-743) provides an efficient way of testing a potentially vastnumber of different substances for ability to modulate activity of apolypeptide. Prior to or as well as being screened for modulation ofactivity, test substances may be screened for ability to interact withthe Rhomboid polypeptide, e.g. in a yeast two-hybrid system (whichrequires that both the polypeptide and the test substance can beexpressed in yeast from encoding nucleic acid). This may be used as acoarse screen prior to testing a substance for actual ability tomodulate protease activity of the polypeptide.

The amount of test substance or compound, which may be added in a methodof the invention, will normally be determined by trial and errordepending upon the type of compound used. Typically, from about 0.01 to100 μM. concentrations of putative inhibitor compound may be used, forexample from 0.1 to 10 μM.

Test compounds may be natural or synthetic chemical compounds used indrug screening programmes. Extracts of plants that contain severalcharacterised or uncharacterised components may also be used.

Methods of the invention may comprise the step of identifying the testcompound as an inhibitor of adhesive micronemal polypeptide cleavage.

One class of putative inhibitor compounds can be derived from theRhomboid polypeptide and/or substrate polypeptide (e.g. adhesivemicronemal polypeptide). Peptide fragments of from 5 to 40 amino acids,for example, from 6 to 10 amino acids may be tested for their ability todisrupt such interaction or activity.

The inhibitory properties of a peptide fragment as described above maybe increased by the addition of one of the following groups to the Cterminal: chloromethyl ketone, aldehyde and boronic acid. These groupsare transition state analogues for serine, cysteine and threonineproteases. The N terminus of a peptide fragment may be blocked withcarbobenzyl to inhibit aminopeptidases and improve stability(Proteolytic Enzymes 2nd Ed, Edited by R. Beynon and J. Bond OxfordUniversity Press 2001).

Antibodies directed to the site of interaction in the protozoan Rhomboidpolypeptide or adhesive micronemal protein form a further class ofputative inhibitor compounds. Candidate inhibitor antibodies may becharacterised and their binding regions determined to provide singlechain antibodies and fragments thereof which are responsible fordisrupting the interaction.

Antibodies may be obtained using techniques that are standard in theart. Methods of producing antibodies include immunising a mammal (e.g.mouse, rat, rabbit, horse, goat, sheep or monkey) with the protein or afragment thereof. Antibodies may be obtained from immunised animalsusing any of a variety of techniques known in the art,.and screened,preferably using binding of antibody to antigen of interest. Forinstance, Western blotting techniques or immunoprecipitation may be used(Armitage et al., 1992, Nature 357: 80-82). Isolation of antibodiesand/or antibody-producing cells from an animal may be accompanied by astep of sacrificing the animal.

As an alternative or supplement to immunising a mammal with a peptide,an antibody specific for a protein may be obtained from a recombinantlyproduced library of expressed immunoglobulin variable domains, e.g.using lambda bacteriophage or filamentous bacteriophage which displayfunctional immunoglobulin binding domains on their surfaces; forinstance see WO92/01047. The library may be naive, that is constructedfrom sequences obtained from an organism which has not been immunisedwith any of the proteins (or fragments), or may be one constructed usingsequences obtained from an organism which has been exposed to theantigen of interest.

Antibodies according to the present invention may be modified in anumber of ways. Indeed, the term “antibody” should be construed ascovering any binding substance having a binding domain with the requiredspecificity. Thus the invention covers antibody fragments, derivatives,functional equivalents and homologues of antibodies, including syntheticmolecules and molecules whose shape mimicks that of an antibody enablingit to bind an antigen or epitope.

Example antibody fragments, capable of binding an antigen or otherbinding partner are the Fab fragment consisting of. the VL, VH, Cl andCHl domains; the Fd fragment consisting of the VH and CHl domains; theFv fragment consisting of the VL and VH domains of a single arm of anantibody; the dAb fragment which consists of a VH domain; isolated CDRregions and F(ab′)2 fragments, a bivalent fragment including two Fabfragments linked by a disulphide bridge at the hinge region. Singlechain Fv fragments are also included.

The reactivities of antibodies on a sample may be determined by anyappropriate means. Tagging with individual reporter molecules is onepossibility. The reporter molecules may directly or indirectly generatedetectable, and preferably measurable, signals. The linkage of reportermolecules may be directly or indirectly, covalently, e.g. via a peptidebond or non-covalently. Linkage via a peptide bond may be as a result ofrecombinant expression of a gene fusion that encodes antibody andreporter molecule. The mode of determining binding is not a feature ofthe present invention and those skilled in the art are able to choose asuitable mode according to their preference and general knowledge.

Antibodies may also be used in purifying and/or isolating Rhomboid andadhesive micronemal polypeptides for use in the present methods, forinstance following production of the polypeptide or peptide byexpression from encoding nucleic acid therefor.

Antibodies may be useful in a therapeutic context (which may includeprophylaxis) to disrupt Rhomboid mediated cleavage of adhesivemicronemal proteins and thus to reduce pathogen invasiveness in thetreatment protozoan infections, including malaria.

Antibodies may also be employed in accordance with the present inventionfor other therapeutic and non-therapeutic purposes which are discussedelsewhere herein.

As described above, adhesive micronemal polypeptide cleavage is anessential step in the infection of host cells by protozoan pathogens.Compounds which inhibit adhesive micronemal polypeptide cleavage maythus be useful in inhibiting protozoan pathogen invasiveness.

Methods of the invention may further comprise the step of determiningthe ability of said test compound to inhibit the invasiveness of aprotozoan pathogen.

This may be achieved by contacting a host cell with a protozoan pathogenin the presence of the test compound under conditions in which thepathogen normally infects the host cell.

Invasiveness of a protozoan pathogen may be determined, for example, inthe presence and absence of test compound, in tissue culture usinghepatocytes or HepG2 cells. Alternatively, the ability of a sporozoiteto glide on a glass slide may be determined. This motility is tightlylinked to invasiveness (Matuschewski K et al EMBO J (2002) 21 (7)1597-1606).

In other assay systems, sporozoites may be injected into an animal model(e.g. rat) to which a test compound is administered and the extent oramount of infection of hepatocytes within the animal determined, andcompared to control animals (Matuschewski K et al supra). Determinationof hepatocyte infection may involve sacrificing and dissecting theanimal model.

A decrease or reduction in the rate of infection in the presencerelative to the absence of test compound is indicative that the testcompound inhibits infectivity.

The test compound may further be isolated and/ormanufactured/synthesised and subsequently formulated into apharmaceutical composition with a pharmaceutically acceptable excipient,vehicle or carrier.

It is desirable that compounds for use in therapeutic contextspreferentially or specifically inhibit protozoan Rhomboid polypeptiderelative to human Rhomboid polypeptide. Methods of the invention mayinclude a further screen to identify such compounds.

A method may therefore comprise the further step of;

-   -   bringing into contact an isolated human Rhomboid polypeptide and        a polypeptide substrate in the presence of the test compound;        and,    -   determining proteolytic cleavage of the substrate by the human        Rhomboid polypeptide.

A human Rhomboid polypeptide may be selected from the group consistingof Human RHBDL-1 (Human Rhomboid-1: Pascall and Brown (1998) FEBS Lett.429, 337-340), Human RHBDL-2 (NM_(—)017821) and Human RHBDL-3.

Suitable polypeptide substrates for human Rhomboids are described aboveand are cleaved by the Rhomboid polypeptide within the transmembranedomain.

A suitable substrate polypeptide may comprise a transmembrane motifwhich has one or more residues of the Drosophila Spitz ASIASGA motifwithin the region proximal to the lumenal or extracellular domain of thepolypeptide (i.e. residues 1 to 8 of the TMD starting at the lumenalboundary) or may comprise a transmembrane motif which none of theresidues of the Drosophila Spitz ASIASGA motif, but which insteadpossess a motif having an equivalent structure which is cleaved byRhomboid polypeptide (e.g. Gurken or other sequence shown in FIG. 1).Preferably a substrate polypeptide comprises one or both of motifsdescribed above in the region proximal to the lumenal or extracellulardomain of the polypeptide.

Following identification of a compound using a method of the inventiondescribed herein, a method may further comprise modifying the compoundto optimise the pharmaceutical properties thereof.

The modification of a ‘lead’ compound identified as biologically activeis a known approach to the development of pharmaceuticals and may bedesirable where the active compound is difficult or expensive tosynthesise or where it is unsuitable for a particular method ofadministration, e.g. peptides are not well suited as active agents fororal compositions as they tend to be quickly degraded by proteases inthe alimentary canal. Modification of a known active compound (forexample, to produce a mimetic) may be used to avoid randomly screeninglarge number of molecules for a target property. Modification of a‘lead’ compound to optimise its pharmaceutical properties commonlycomprises several steps. Firstly, the particular parts of the compoundthat are critical and/or important in determining the target propertyare determined. In the case of a peptide, this can be done bysystematically varying the amino acid residues in the peptide, e.g. bysubstituting each residue in turn. These parts or residues constitutingthe active region of the compound are known as its “pharmacophore”.

Once the pharmacophore has been found, its structure is modelledaccording to its physical properties, e.g. stereochemistry, bonding,size and/or charge, using data from a range of sources, e.g.spectroscopic techniques, X-ray diffraction data and NMR.

Computational analysis, similarity mapping (which models the chargeand/or volume of a pharmacophore, rather than the bonding between atoms)and other techniques can be used in this modelling process.

In a variant of this approach, the three-dimensional structure of theligand and its binding partner are modelled. This can be especiallyuseful where the ligand and/or binding partner change conformation onbinding, allowing the model to take account of this in the optimisationof the lead compound.

A template molecule is then selected onto which chemical groups thatmimic the pharmacophore can be grafted. The template molecule and thechemical groups grafted on to it can conveniently be selected so thatthe modified compound is easy to synthesise, is likely to bepharmacologically acceptable, and does not degrade in vivo, whileretaining the biological activity of the lead compound. The modifiedcompounds found by this approach can then be screened to see whetherthey have the target property, or to what extent they exhibit it.Modified compounds include mimetics of the lead compound.

Further optimisation or modification can then be carried out to arriveat one or more final compounds for in vivo or clinical testing.

Rhomboid and adhesive miconemal polypeptides may also be used in methodsof designing mimetics which are suitable for inhibiting protozoanpathogen infectivity.

The present invention provides a method of designing mimetics having thebiological activity of inhibiting the Rhomboid mediated cleavage ofadhesive miconemal polypeptides and thus inhibiting protozoan pathogeninvasiveness, said method comprising:

(i) analysing a compound having the biological activity to determine theamino acid residues essential and important for the activity to define apharmacophore; and,

(ii) modelling the pharmacophore to design and/or screen candidatemimetics having the biological activity.

A suitable compound may be, for example, a protozoan Rhomboidpolypeptide or fragment as described herein.

Suitable modelling techniques are known in the art. This includes thedesign of so-called “mimetics” which involves the study of thefunctional interactions of the molecules and the design of compoundswhich contain functional groups arranged in such a manner that theycould reproduced those interactions.

The modelling and modification of a ‘lead’ compound to optimise itsproperties, including the production of mimetics, is described above.

The activity or function of a protozoan Rhomboid polypeptide may beinhibited, as noted, by means of a compound that interferes in some waywith the interaction of protozoan Rhomboid with adhesive micronemalpolypeptides or other suitable substrate polypeptides. An alternativeapproach to inhibition employs regulation at the nucleic acid level toinhibit activity or function by down-regulating production of protozoanRhomboid.

For instance, expression of a gene may be inhibited using anti-sense orRNAi technology. The use of these approaches to down-regulate geneexpression is now well-established in the art.

Anti-sense oligonucleotides may be designed to hybridise to thecomplementary sequence of nucleic acid, pre-mRNA or mature mRNA,interfering with the production of Rhomboid polypeptide so that itsexpression is reduced or completely or substantially completelyprevented. In addition to targeting coding sequence, antisensetechniques may be used to target control sequences of a gene, e.g. inthe 5′ flanking sequence, whereby the antisense oligonucleotides caninterfere with expression control sequences. The construction ofantisense sequences and their use is described for example in Peyman andUlman, Chemical Reviews, 90: 543-584, (1990) and Crooke, Ann. Rev.Pharmacol. Toxicol., 32: 329-376, (1992).

Oligonucleotides may be generated in vitro or ex vivo for administrationor anti-sense RNA may be generated in vivo within cells in whichdown-regulation is desired. Thus, double-stranded DNA may be placedunder the control of a promoter in a “reverse orientation” such thattranscription of the anti-sense strand of the DNA yields RNA which iscomplementary to normal mRNA transcribed from the sense strand of thetarget gene. The complementary anti-sense RNA sequence is thought thento bind with mRNA to form a duplex, inhibiting translation of theendogenous mRNA from the target gene into protein. Whether or not thisis the actual mode of action is still uncertain. However, it isestablished fact that the technique works.

The complete sequence corresponding to the coding sequence in reverseorientation need not be used. For example fragments of sufficient lengthmay be used. It is a routine matter for the person skilled in the art toscreen fragments of various sizes and from various parts of the codingor flanking sequences of a gene to optimise the level of anti-senseinhibition. It may be advantageous to include the initiating methionineATG codon, and perhaps one or more nucleotides upstream of theinitiating codon. A suitable fragment may have about 14-23 nucleotides,e.g. about 15, 16 or 17.

An alternative to anti-sense is to use a copy of all or part of thetarget gene inserted in sense, that is the same, orientation as thetarget gene, to achieve reduction in expression of the target gene byco-suppression; Angell & Baulcombe (1997) The EMBO Journal 16, 12:3675-3684; and Voinnet & Baulcombe (1997) Nature 389: pg 553). Doublestranded RNA (dsRNA) has been found to be even more effective in genesilencing than both sense or antisense strands alone (Fire A. et alNature, 391, (1998)) and has been used effectively in Plasmodium(Malhotra P et al Mol. Microb. (2002) 45(5) 1245-1254; McRobert L et alMol. Biochem. Parasitol. (2002) 119(2) 273-278). dsRNA mediatedsilencing is gene specific and is often termed RNA interference (RNAi).

RNA interference is a two step process. First, dsRNA is cleaved withinthe cell to yield short interfering RNAs (siRNAs) of about 21-23 ntlength with 5′ terminal phosphate and 3′ short overhangs (˜2 nt). ThesiRNAs target the corresponding mRNA sequence specifically fordestruction (Zamore P. D. Nature Structural Biology, 8, 9, 746-750,(2001)

RNAi may be also be efficiently induced using chemically synthesizedsiRNA duplexes of the same structure with 3′-overhang ends (Zamore P Det al Cell, 101, 25-33, (2000)). Synthetic siRNA duplexes have beenshown to specifically suppress expression of endogenous andheterologeous genes in a wide range of mammalian cell lines (Elbashir SM. et al. Nature, 411, 494-498, (2001)).

Another possibility is that nucleic acid is used which on transcriptionproduces a ribozyme, able to cut nucleic acid at a specific site—thusalso useful in influencing gene expression. Background references forribozymes include Kashani-Sabet and Scanlon, 1995, Cancer Gene Therapy,2(3): 213-223, and Mercola and Cohen, 1995, Cancer Gene Therapy, 2(1),47-59.

Thus, a modulator of protozoan Rhomboid activity and thus a modulator ofprotozoan pathogen infectivity may comprise a nucleic acid moleculecomprising all or part of a Rhomboid coding sequence shown in Table 1 orthe complement thereof

Such a molecule may suppress the expression of protozoan Rhomboidpolypeptide and may comprise a sense or anti-sense Rhomboid codingsequence or may be a Rhomboid specific ribozyme, according to the typeof suppression to be employed.

The type of suppression will also determine whether the molecule isdouble or single stranded and whether it is RNA or DNA.

Another aspect of the present invention provides a method of producing apharmaceutical composition comprising;

-   -   identifying a compound which inhibits the invasiveness of a        protozoan pathogen using a method described herein; and,    -   admixing the compound identified thereby with a pharmaceutically        acceptable carrier.

As described above, a method of the invention may comprise the step ofmodifying the compound to optimise the pharmaceutical propertiesthereof.

Another aspect of the invention provides a method for preparing apharmaceutical composition for treating a protozoan pathogen infectioncomprising;

-   -   i) identifying a compound which modulates the proteolytic        activity of a Rhomboid polypeptide,    -   ii) synthesising the identified compound, and;    -   iii) incorporating the compound into a pharmaceutical        composition.

The identified compound may be synthesised using conventional chemicalsynthesis methodologies. Methods for the development and optimisation ofsynthetic routes are well known to a skilled person.

The compound may be modified and/or optimised as described above.

Incorporating the compound into a pharmaceutical composition may includeadmixing the synthesised compound with a pharmaceutically acceptablecarrier or excipient.

Another aspect of the invention provides a compound which modulatesprotozoan pathogen infectivity obtained by a method as described herein.Such a compound may comprise or consist of a peptide fragment of aprotozoan Rhomboid polypeptide.

Another aspect of the invention provides a pharmaceutical compositioncomprising a compound which modulates the proteolytic activity of aRhomboid polypeptide obtained by a method described herein.

In other aspects the invention provides the use of a compound obtainedby a method described herein in the manufacture of a composition fortreatment of a protozoan pathogen infection and a method comprisingadministration of a composition obtained by a method described herein toa patient for treatment of a protozoan pathogen infection.

Protozoan pathogens are described above. Disorders associated withinfection with a protozoan pathogen include malaria, toxoplasmosis,cryptosporidosis, diarrhoea associated with Isospora belli or Cyclosporacayetanis infection, and various livestock disorders associated withEimeria infection.

Whether it is a polypeptide, antibody, peptide, anti-sense, sense orsiRNA nucleic acid molecule, small molecule, mimetic or otherpharmaceutically useful compound according to the present invention thatis to be given to an individual, administration is preferably in a“prophylactically effective amount” or a “therapeutically effectiveamount” (as the case may be, although prophylaxis may be consideredtherapy), this being sufficient to show benefit to the individual. Theactual amount administered, and rate and time-course of administration,will depend on the nature and severity of what is being treated.Prescription of treatment, e.g. decisions on dosage etc, is within theresponsibility of general practitioners and other medical doctors.

A composition may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated.

Pharmaceutical compositions according to the present invention, and foruse in accordance with the present invention, may include, in additionto active ingredient, a pharmaceutically acceptable excipient, carrier,buffer, stabiliser or other materials well known to those skilled in theart. Such materials should be non-toxic and should not interfere withthe efficacy of the active ingredient. The precise nature of the carrieror other material will depend on the route of administration, which maybe oral, or by injection, e.g. cutaneous, subcutaneous or intravenous.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may include a solid carriersuch as gelatin or an adjuvant. Liquid pharmaceutical compositionsgenerally include a liquid carrier such as water, petroleum, animal orvegetable oils, mineral oil or synthetic oil. Physiological salinesolution, dextrose or other saccharide solution or glycols such asethylene glycol, propylene glycol or polyethylene glycol may beincluded.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the active ingredient will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as Sodium Chloride. Injection, Ringer'sInjection, Lactated Ringer's Injection. Preservatives, stabilisers,buffers, antioxidants and/or other additives may be included, asrequired.

Liposomes, particularly cationic liposomes, may be used in carrierformulations.

Examples of techniques and protocols mentioned above can be found inRemington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

Another aspect of the invention provides a method of identifying aprotozoan Rhomboid polypeptide comprising,

-   -   (a) providing a test Rhomboid polypeptide,    -   (b) bringing into contact a substrate polypeptide and the test        Rhomboid polypeptide under conditions in which the substrate        polypeptide is normally proteolytically cleaved; and,    -   (c) determining cleavage of the substrate polypeptide.

A suitable test Rhomboid polypeptide may comprise an amino acid sequenceencoded by a nucleic acid sequence shown in Table 1. Screening databasesmay identify other suitable test Rhomboid polypeptides, in particular,databases of protozoan nucleic acid sequence, using the bioinformaticstechniques discussed above.

Suitable substrate polypeptides include protozoan adhesive micronemalpolypeptides. A suitable adhesive micronemal polypeptide may belong tothe thrombospondin-related-anonymous proteins (TRAPs) family. Examplesof suitable polypeptides include AMA1, MIC2, MIC6, MIC8, MIC12, (from T.gondii), TRAP, CTRP (from P. falciparum and P. berghei), MIC1 and MIC4(from E. tenella) or fragments thereof which comprise the transmembranedomain. In particular, CTRP and/or AMA1 may be used.

Other aspects of the invention relate to protozoan Rhomboid polypeptidesand encoding nucleic acids.

An aspect of the invention provides a protozoan Rhomboid polypeptidewhich proteolytically cleaves the transmembrane domain of an adhesivemicronemal polypeptide.

Such a polypeptide,may have a sequence encoded by a nucleic acidsequence shown in Table 1 (for example Pf Rhol-7) or may be a fragmentof such a sequence which consists of fewer residues than the full-lengthprotozoan Rhomboid polypeptide.

The KDEL ER retention sequence is not found in natural protozoanRhomboid polypeptides and directs the expressed Rhomboid polypeptide tobe retained the ER (endoplasmic reticulum) rather than the miconemes ofthe protozoan cell. As described below, Rhomboid polypeptides labelledwith an ER retention sequence such as KDEL may be particularly useful incertain embodiments of the invention, as proteolyic cleavage by suchpolypeptides avoids potential problems with variations in secretionefficiency.

An aspect of the invention thus provides an isolated protozoan Rhomboidpolypeptide as described herein comprising a C terminal ER retentionsequence. A suitable retention sequence consists of the amino acidsequence KDEL.

Another aspect of the invention provides an isolated nucleic acidencoding a protozoan Rhomboid polypeptide as described above.

The coding sequence may be a nucleic acid sequence listed in Table 1 orit may be a mutant, variant, derivative or allele of a sequence listed.The sequence may differ from a sequence of Table 1 by a change, which isone or more of addition, insertion, deletion and substitution of one ormore nucleotides of the sequence shown. Changes to a nucleotide sequencemay result in an amino acid change at the protein level, or not, asdetermined by the genetic code.

Thus, nucleic acid according to the present invention may include asequence different from a sequence listed in Table 1 yet encode apolypeptide with the same amino acid sequence. As described above, codonusage may be adjusted in order to express an amino acid sequence in aparticular host system, such as a mammalian cell.

An isolated nucleic acid may share greater than about 10% sequenceidentity with a nucleic acid sequence of Table 1 greater than 20%,greater than 30%, greater than 40%, greater than 50%, greater than 60%,greater than about 70%, greater than about 80%, greater than about 90%,or greater than about 95%.

The present invention also extends to nucleic acid that hybridizes witha sequence listed in Table 1 under stringent conditions. Suitableconditions include, e.g. for sequences that are about 80-90% identical;hybridisation overnight at 42° C. in 0.25 M Na₂HPO₄, pH 7.2, 6.5% SDS,10% dextran sulfate and a final wash at 55° C. in 0.1×SSC, 0.1% SDS. Forsequences that are greater than about 90% identical, suitable conditionsinclude. hybridization overnight at 65° C. in 0.25M Na₂HPO₄, pH 7.2,6.5% SDS, 10% dextran sulfate and a final wash at 60° C. in 0.1×SSC,0.1% SDS.

A convenient way of producing a polypeptide for use in assays andmethods according to the present invention is to express nucleic acidencoding it, by use of the nucleic acid in an expression system.Accordingly, the present invention also encompasses a method of making apolypeptide (as disclosed), the method including expression from nucleicacid encoding the polypeptide and testing for Rhomboid proteaseactivity. This may conveniently be achieved by growing a host cell inculture, containing such a vector, under appropriate conditions thatcause or allow expression of the polypeptide. Polypeptides may also beexpressed in in vitro systems, such as reticulocyte lysate.

Another aspect of the present invention therefore provides a method ofproducing a Rhomboid polypeptide comprising:

-   -   (a) causing expression from nucleic acid which encodes a        protozoan Rhomboid polypeptide in a suitable expression system        to produce the polypeptide recombinantly;    -   (b) testing the recombinantly produced polypeptide for Rhomboid        protease activity.

Suitable nucleic acid sequences include a nucleic acid sequence encodinga protozoan rhomboid polypeptide a mutant, variant or allele thereof asdescribed herein.

A polypeptide may be isolated and/or purified (e.g. using an antibody)for instance after production by expression from encoding nucleic acid(for which see below). Thus, a polypeptide may be provided free orsubstantially free from contaminants with which it is naturallyassociated (if it is a naturally-occurring polypeptide). A polypeptidemay be provided free or substantially free of other polypeptides.

The recombinantly produced polypeptide may be isolated and/or tested forRhomboid protease activity by determination of the cleavage of asubstrate polypeptide such as an adhesive micronemal polypeptide uponincubation of the polypeptide with the substrate polypeptide.

An isolated nucleic acid as described herein, for example a nucleic acidencoding a protozoan Rhomboid polypeptide, may be comprised in a vector.Such a vector may further include a nucleic acid sequence encoding asubstrate polypeptide such as a protozoan adhesive micronemal protein.Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorfragments, polyadenylation sequences, enhancer sequences, marker genesand other sequences as appropriate. Vectors may be plasmids, viral e.g.'phage, or phagemid, as appropriate. For further details see, forexample, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrooket al., 1989, Cold Spring Harbor Laboratory Press. Many known techniquesand protocols for manipulation of nucleic acid, for example inpreparation of nucleic acid constructs, mutagenesis, sequencing,introduction of DNA into cells and gene expression, and analysis ofproteins, are described in detail in Current Protocols in MolecularBiology, Ausubel et al. eds., John Wiley & Sons, 1992.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known. Suitable host cells includebacteria, eukaryotic cells such as mammalian and yeast, baculovirus andprotozoan systems. Mammalian cell lines available in the art forexpression of a heterologous polypeptide include Chinese hamster ovarycells, HeLa cells, baby hamster kidney cells, COS cells and many others.A common, preferred bacterial host is E. coli.

Further aspects of the invention provide a host cell containingheterologous nucleic acid encoding a protozoan Rhomboid polypeptide,including a Rhomboid polypeptide which has a KDEL tag or which is afragment of a full length Rhomboid sequence, and a host cell containingheterologous nucleic acid encoding a protozoan Rhomboid polypeptide anda substrate polypeptide, for example a protozoan adhesive micronemalpolypeptide. Nucleic acid encoding the protozoan Rhomboid polypeptideand substrate polypeptide may be present on a single nucleic acidconstruct or vector within the host cell or nucleic acid encoding thetwo polypeptides may be present on separate constructs or vectors.

The nucleic acid may be integrated into the genome (e.g. chromosome) ofthe host cell. Integration may be promoted by inclusion of sequencesthat promote recombination with the genome, in accordance with standardtechniques. The nucleic acid may be on an extra-chromosomal vectorwithin the cell.

The introduction of nucleic acid into a host cell, which may(particularly for in vitro introduction) be generally referred towithout limitation as “transformation”, may employ any availabletechnique. For eukaryotic cells, suitable techniques may include calciumphosphate transfection, DEAE-Dextran, electroporation, liposome-mediatedtransfection and transduction using retrovirus or other virus, e.g.vaccinia or, for insect cells, baculovirus. For bacterial cells,suitable techniques may include calcium chloride transformation,electroporation and transfection using bacteriophage.

Marker genes such as antibiotic resistance or sensitivity genes may beused in identifying clones containing nucleic acid of interest, as iswell known in the art.

The introduction may be followed by causing or allowing expression fromthe nucleic acid, e.g. by culturing host cells (which may include cellsactually transformed although more likely the cells will be descendantsof the transformed cells) under conditions for expression of the gene,so that the encoded polypeptide is produced.

A Rhomboid polypeptide may be co-expressed in a host cell with asubstrate polypeptide, such as a protozoan adhesive micronemalpolypeptide, and the Rhomboid serine protease activity determined bydetermining cleavage of the substrate polypeptide. Cleavage may bedetermined by determining the presence or absence of soluble cleavageproducts which may be secreted into the culture medium.

Aspects of the present invention will now be illustrated with referenceto the following experimental exemplification, by way of example and notlimitation. Further aspects and embodiments will be apparent to those ofordinary skill in the art. All documents mentioned in this specificationare hereby incorporated herein by reference.

FIG. 1 shows an alignment of transmembrane domains of micronemalproteins from apicomplexan species with Drosophila Spitz and Keren. Allare single-pass type 1 transmembrane proteins. Black lines indicate thepredicted TMD regions: the line above the sequences predicts themicronemal protein TMDs; the line below predicts the TMDs of Spitz andKeren. The GA or GG motif approximately seven residues into the TMD(double underline) and the conserved small residues near theluminal/extracellular face of the TMD (single underline) are essentialfor rhomboid cleavage of Spitz and are underscored. Note also theconserved tyrosine (Y) at the cytoplasmic face of the TMDs. Accessionnumbers of the sequences are shown in Table 2.

Table 1a shows examples of Plasmodium falciparum rhomboids which wereinitially identified by the Pfam motif-finding algorithm, with a scoreof greater than 10. Four annotations are listed: sequencing consortiumannotation (Sanger), plus 3 automatic gene prediction algorithms,FullPhat, GlimmerM and Genefinder. The approximate position on thechromosome is also provided along with the predicted sequence around therhomboid active site. Rhomboids 2 and 5 are predicted to bemitochondrial, based on the existence of a mitochondrial targetingsequence predicted by MitoProt or Predotar. These genes represent themost reliable prediction of true rhomboids in the published P.falciparum genome sequence as predicted by the Pfam motif-findingalgorithm, with a score of greater than 10.

Table 1b shows an example of a candidate Toxoplasma gondii rhomboid(ref: http://ParaDB.cis.upenn.edu/toxo/index.html)

Table 2 shows examples of substrates for rhomboid proteases fromapicomplexan species.

EXPERIMENTAL

Identification of Plasmodium Rhomboid Polypeptides.

The published Plasmodium genome sequence was searched for Rhomboidspolypeptides using PFAM as described above, selecting scores higher than10, followed by visual inspection to identify key Rhomboid residues.

A number of putative Rhomboids were identified using PFAM. These werefurther analysed for the presence of residues equivalent to residuesN169, G215, S217 and H281 of Drosophila Rhomboid-1, which are requiredfor catalytic activity. Candidates identified as being within thesubclass of mitochondrial rhomboids were also excluded by prediction ofthe existence of a mitohondrial targeting sequence with PREDOTARalgorithm (www.inra.fr/predotar) or MitoProt (Claros M., et al Eur. J.Biochem. (1996) 241 779-786)

Rhomboid polypeptides identified by this approach are listed in Table 1.

Cloning of Plasmodium Rhomboid

Rhomboid coding sequences were amplified by PCR from Plasmodium cDNAusing the protocols set out in Sambrook & Russell Molecular Cloning, ALaboratory Manual, 3^(rd) Edition, Cold Spring Harbor Laboratory Press,2001, and Ausubel et al, Short Protocols in Molecular Biology, JohnWiley and Sons, 1992. The amplified sequences were then inserted into apcDNA3.1 (Invitrogen).

The Pf Rho 1, 3, 4 and 7 genes were resynthesised (Geneart, DE) withcodon usage adjusted as described in Kocken et al (2002) Infect Immun70(8) 4471-4476 to optimise expression in mammalian cell lines.

Expression of Pf rhomboids-1, -3 and -7 was observed in COS cells.

Cloning of Adhesive Micronemal Proteins of Plasmodium

Oligonucleotide primers for amplification of the adhesive micronemalproteins Ama-1 and CTRP of Plasmodium were designed based on thepublished sequences using conventional primer design software.

The coding sequences were amplified using PCR in accordance withstandard techniques, and cloned into a standard mammalian expressionvector for expression in cell culture. The coding sequences incorporateda triple-HA tag to facilitate detection. Expression was detected byconventional Western blotting techniques using available anti-HAantibodies (Roche).

Plasmodium Rhomboid Activity

Plasmodium rhomboid activity was determined using published methodsdescribed in Urban et al Cell (2001) 107(2): 173-82 and WO02/093177.

Briefly, Pf rhomboids-1 and -3 were co-expressed with GFP-tagged Spitzunder control of a CMV promoter; as follows; COS cells were grown inDMEM medium (supplemented with 10% foetal calf serum), and transfectedwith FuGENE 6 Transfection Reagent (Roche).

Cells were co-transfected in 35mm culture wells with 25-250 ng of (a)rhomboid construct comprising the Pf-rhomboid-1 or -3 coding sequenceinserted into the vector pcDNA 3.1+ (Invitrogen), and (b) substrateconstruct comprising the GFP-Spitz coding sequence inserted into pcDNA3.1+ (Invitrogen).

Empty plasmid was used to bring the total DNA to 1 μg per well.

Construct (b) was transfected into COS cells in the absence of construct(a) as a control for endogenous cleavage of the substrate.

24-30 hours post-transfection, the medium was replaced with serum-freemedium; this was harvested 24 hours later and cells were lysed inSDS-sample buffer.

For some experiments, the serum-free medium was supplemented with themetalloprotease inhibitor batimastat (British Biotech) or ilomostat(Calbiochem) to minimize endogenous substrate cleavage in the assay. Forinhibitor assays, a test compound may be included in the serum-freemedium.

Spitz cleavage was assayed by detecting the accumulation of theGFP-tagged Spitz extracellular domain in the medium by standard westernblotting techniques, using an anti-GFP antibody(Santa Cruz Biologicals).

Parallel experiments were also performed using a C-terminally taggedform of Spitz. Cleavage of the C-terminal tagged Spitz was assayed bydetecting the intracellular cleaved product in western blots of cellextracts.

Both Pf rhomboid-1 and -3 were observed to show specific cleavage ofSpitz, which was not inhibited by batimastat. Batimastat is a potentinhibitor of metalloproteases that can cause non-specific shedding ofcell surface proteins. These results show that both Pf rhomboid-1 and -3are active rhomboid proteases that can recognise ‘Spitz-like’ TMDs.

Cleavage of Pf adhesion protein Ama-1

Pf adhesion protein Ama-1 was recoded with a mammalian codon usageprofile to allow for expression in mammalian cells.

Cleavage of Ama-1 by Drosophila Rhomboid-1 was determined using themethod'set out above. Briefly, Ama-1 and Drosophila Rhomboid-1 wereco-expressed as described above in COS cells using the CMV promoter forboth constructs. Standard western blotting techniques using ananti-Ama-1 antibody were employed to detect the accumulation of theextracellular domain of cleaved Ama-1 in the COS cell medium.

Efficient cleavage of Ama-1 by Drosophila Rhomboid-1 was observed. Thiscleavage was not sensitive to batimastat. This shows that Ama-1 has arhomboid-cleavable TMD.

Cleavage of Pf Adhesion Protein CTRP

Cleavage of the CTRP TMD by Drosophila Rhomboid-1 was also determinedusing the methods described above.

Briefly, Drosophila Rhomboid-1 was co-expressed as described above inCOS cells with a chimeric protein comprising (from N- to C-terminus) thesignal peptide of Spitz, followed by GFP, followed by the Pf CTRPtransmembrane domain, followed by the cytoplasmic domain of TGFalpha(i.e. the CTRP TMD with heterologous extracellular and cytoplasmicdomains). The CMV promoter was used to express both constructs.

Cleavage of the CTRP TMD was assayed by the standard technique describedabove. If the CTRP TMD is cleaved by the Rhomboid protein, GFPaccumulates in the medium of the transfected cells in abatimastat-insensitive manner. Accumulated GFP is then detected with ananti-GFP antibody (Santa Cruz Biologicals) as described above.

It was observed that the TMD from Pf adhesion protein CTRP is cleavedefficiently by Drosophila rhomboid-1.

Expression of Rhomboid in Plasmodium

Pairs of amplification primers specific for each P. falciparum Rhomboidwere designed using conventional primer design software. The expressionof Plasmodium Rhomboid proteins was analysed by performing PCR on cDNAprepared from RNA isolated from the blood stage of Plasmodium falciparumusing standard techniques.

Rhomboids-1, -3, -4 and -6 are all observed to be expressed in bloodstage Plasmodium parasites.

Plasmodium Rhomboids are therefore present at the blood cell invasionstage of the parasitic life-cycle and may play a key role in mediatingthis invasion.

In Vivo Role of Rhomboids in Infectivity of Parasites

Mutant strains of P. falciparum are generated in which the candidaterhomboid protein is knocked out. The infectiousness of the mutantparasite is then determined. The knockout is done either by standardtargeted gene disruption (Sultan et al Cell 90 511) or, moreconveniently, by RNA interference (RNAi), as described below.

100 μpg of RNA corresponding to each candidate Rhomboid gene issynthesized by in vitro transcription from 5 μg linearized plasmidtemplates according to manufacturer's instructions (Promega Ribomaxsystem). The resulting RNA is purified using the RNeasy protocol(Qiagen), denatured by boiling, and annealed in 1 mM Tris-HCl pH7.4, 1mM EDTA overnight. The resulting dsRNA is then added to a culture of P.falciparum sporozoites (Malhotra et al Mol. Microb. 45 1245-1254).

The reduced infectivity or gliding behaviour of the knockout parasitesrelative to the wild-type controls confirms that rhomboid regulates theinfectious process. Impairment of the proteolysis of the adhesivemicronemal proteins may also be established in the rhomboid mutant (orRNAi) cells, by determining the production of cleaved product by Westernblotting using anti-TRAP antibodies (Sharma et al Infection & Immunity64 2172-2179, Gantt et al Infection & Immunity 68 3667-3673). TABLE 1aApprox anger FullPhat GlimmerM Genefinder Chromosome location MotifRhomboid FE03040c chr5.phat_92 chr5.glm_74 chr5.gen_274 5 289 kb GSSGRho1 FE0755c chr5.phat_172 chr5.glm_161 chr5.gen_236 5 624 kb GASG Rho2(mit

F11_0150 chr11.phat_164 chr11.glm_163 Not predicted 11 538 kb GAST Rho3F13_0312 chr13.phat_576 chr13.glm_633 chr13.gen_395 13 2.22 Mb GSSS Rho5(mit

F13_0241 chr13.phat_452 chr13.glm_494 chr13.gen_456 13 1.73 Mb GASG Rho4F14_0110 chr14.phat_108 Not predicted chr14.gen_705 14 453 kb SSSS Rho6(?) AL8P1.16 chr8.phat_47 chr8.glm_34 chr8.gen_290 8 135 kb GAST Rho7

TABLE 1b Toxoplasma gondii Genbank consensus ID accession Motif RhomboidCtoxoqual_3360 AA531635 GAST TgRho1

TABLE 2 Accession Protein Species no. PfTRAP Plasmodium U67764falciparum PfCTRP Plasmodium U34363 falciparum PfAMA1 PlasmodiumAF277003 falciparum EBL-175 Plasmodium AAM33518 falciparum MAEBLPlasmodium AY042084 falciparum TgMIC2 Toxoplasma TGU62660 gondii TgMIC6Toxoplasma AF110270 gondii TgMIC12 Toxoplasma Opitz et gondii al. TgAMA1Toxoplasma AF010264 gondii PbTRAP Plasmodium U67763 berghei EtMIC1Eimeria AF032905 tenella EtMIC4 Eimeria CAC34726 tenella Sm70Sarcocystis AAK35069 muris

1. A method for identifying and/or obtaining a compound which inhibitsinfectivity of a protozoan pathogen, which method comprises: (a)contacting an isolated Rhomboid polypeptide and an isolated substratepolypeptide in the presence of a test compound; and (b) determiningproteolytic cleavage of the substrate protein.
 2. A method according toclaim 1 wherein the protozoan pathogen is an apicomplexan pathogen.
 3. Amethod according to claim 2 wherein the apicomplexan pathogen isselected from the group consisting of Plasmodium, Toxoplasma, Eimeria,Sarcocystis, Cyclospora, Isospora, Cryptosporidium, Babesia andTheileria.
 4. A method according to claim 1 wherein the Rhomboidpolypeptide is a protozoan Rhomboid protein.
 5. A method according toclaim 4 wherein the Rhomboid polypeptide is encoded by a nucleic acidsequence shown in Table
 1. 6. A method according to claim 1 wherein thesubstrate polypeptide comprises a lumenal domain and a TMD, the TMDhaving a region proximal to the lumenal domain which comprises one ormore of residues 138-144 of the Drosophila Spitz sequence (SEQ ID NO:1,ASIASGA).
 7. A method according to claim 6 wherein the substratepolypeptide comprises a TMD and a lumenal domain, the TMD having aregion proximal to a lumenal domain which has the sequence of residues138-144 of Drosophila Spitz.
 8. A method according to claim 6 whereinthe substrate polypeptide is an adhesive micronemal polypeptide.
 9. Amethod according to claim 8 wherein the substrate polypeptide is encodedby a nucleic acid sequence shown in Table
 2. 10. A method according toclaim 9 wherein the substrate polypeptide is Ama-1 or CTRP.
 11. A methodaccording to any one of the preceding claims wherein the substratepolypeptide and the Rhomboid polypeptide comprise ER (endoplasmicreticulum) retention signals.
 12. A method according to claim 10 whereinthe endoplasmic reticulum retention signals are (SEQ ID NO:2) KDEL or KK Xaa Xaa.
 13. A method according to claim 1 wherein the substratepolypeptide comprises an extracellular domain having a detectable label.14. A method according to claim 13 wherein the detectable label is GFP.15. A method according to claim 1 wherein said Rhomboid polypeptide andsaid substrate polypeptide are expressed in a host cell fromheterogeneous nucleic acid.
 16. A method according to claim 1 comprisingthe further step of; (c) bringing into contact an isolated humanRhomboid polypeptide and a polypeptide substrate in the presence of thetest compound; and, (d) determining proteolytic cleavage of thesubstrate by the human Rhomboid polypeptide.
 17. A method according toclaim 1 comprising identifying said test compound as a modulator ofadhesive micronemal polypeptide cleavage.
 18. A method according toclaim 17 further comprising determining the ability of said testcompound to inhibit the invasiveness of a protozoan pathogen.
 19. Amethod according to claim 17 comprising isolating said test compound.20. A method according to claim 19 comprising synthesising the testcompound.
 21. A method according to claim 19 comprising modifying thetest compound to optimise its pharmacological properties.
 22. A methodaccording to claim 17 comprising formulating said test compound in apharmaceutical composition with a pharmaceutically acceptable excipient,vehicle or carrier.
 23. A compound which modulates protozoan pathogeninfectivity obtained by a method of claim
 1. 24. A compound according toclaim 23 comprising a peptide fragment of a protozoan Rhomboidpolypeptide.
 25. A method of producing a pharmaceutical compositioncomprising; identifying a compound which inhibits the infectivity of aprotozoan pathogen using a method according to claim 1; and, admixingthe compound identified thereby with a pharmaceutically acceptablecarrier.
 26. A method according to claim 25 comprising the step ofmodifying the compound to optimise the pharmaceutical propertiesthereof.
 27. A method for preparing a pharmaceutical composition fortreating a protozoan pathogen infection comprising; i) identifying acompound which modulates the proteolytic activity of a Rhomboidpolypeptide, ii) synthesising the identified compound, and; iii)incorporating the compound into a pharmaceutical composition.
 28. Apharmaceutical composition comprising a compound according to claim 23.29. Use of a compound according to claim 23 in the manufacture of acomposition for treatment of a protozoan pathogen infection.
 30. Amethod comprising administration of a composition according to claim 23to a patient for treatment of a protozoan pathogen infection.
 31. Amethod according to claim 30 wherein the protozoan pathogen is anapicomplexan pathogen selected from the group consisting of Plasmodium,Babesia, Theileria, Toxoplasma, Eimeria, Sarcocystis, Cyclospora,Isospora and Cryptosporidium.
 32. A method of identifying a protozoanRhomboid polypeptide comprising; (a) providing a test protozoan Rhomboidpolypeptide, (b) bringing into contact a substrate polypeptide and thetest Rhomboid polypeptide under conditions in which the substratepolypeptide is normally proteolytically cleaved; and, (c) determiningcleavage of the substrate polypeptide.
 33. A method according to claim32 wherein the test Rhomboid polypeptide comprises an amino acidsequence encoded by a nucleic acid sequence shown in Table
 1. 34. Amethod according to claim 32 wherein the substrate polypeptide comprisesthe lumenal region, of the TMD of Spitz, Gurken, Keren, Ama-1 or CTRP.35. A method according to claim 32 wherein the substrate polypeptidecomprises an amino acid sequence encoded by a nucleic acid sequenceshown in Table
 2. 36. A method according to claim 35 wherein thesubstrate polypeptide is Ama-1 or CTRP.